The Importance of Tissue Blocking in IHC and IF

When working in a histology lab, having an antibody with a high specificity for your target antigen is critical. However, even if you have the perfect antibody, you may still run into problems with Autofluorescence and non-specific binding to the tissue. This problem is typically due to the presence of non-specific binding sites within the tissue and is easily rectifiable through tissue blocking prior to antibody labeling.

Rehydrated Kidney Sample

Tissue Blocking

Tissue blocking is the application of a blocking agent, typically the blood serum of an unrelated animal such as bovine, horse, or donkey, to a tissue to eliminate the non-specific binding of our antibodies, in turn reducing Autofluorescence. The blood serum or other blocking agent does this by binding to the non-specific binding sites on the tissue, blocking them so that our antibodies can not attach to them. Without tissue blocking, it is nearly impossible to tell what is true labeling versus Autofluorescence of the tissue. Common interactions that can cause non-specific antibody binding are adsorption, attachment due to differences in charge, and attachment due to hydrophobic or hydrophilic properties.

Types of Tissue Blocking

Protein Blocking

Protein blocking is common in all IHC and IF protocols, and typically uses normal bovine serum or normal donkey serum to block free non-specific binding sites on tissue. Typically, this involves adding a diluted solution of normal serum to the sample slides and incubating them at ambient temperature for 30 minutes to one hour. The serum used should originate from the same species that the secondary antibodies come from, ensuring that the secondary antibodies do not bind to the blocking serum.

Protein blocking is especially important in cases where the antibody is from the same host species as the tissue being labeled. An example of this would be using a mouse CD3 antibody to label mouse tissue. In this case the mouse antibody will bind to endogenous Fc and IgG receptors on the mouse tissue, causing non-specific binding and Autofluorescence. To avoid this, chose your antibodies carefully and do not choose to use a mouse antibody on mouse tissue. If there is no other choice of antibody available, an additional mouse-on-mouse blocking step can be added to the protocol to block free antibody binding sites on the tissue and limit Autofluorescence.

Endogenous Enzyme Blocking

Some fluorophores, like peroxidase and alkaline phosphatase, are naturally occurring in tissue. This can cause high background staining and makes the interpretation of true labeling quite difficult. Peroxidases are commonly found in kidney, liver, and red blood cells, making the staining of these tissues using horseradish peroxidase (HRP) tagged antibodies difficult. In this case, we can check for endogenous peroxidase activity by using DAB substrate prior to primary antibody application. If the tissue turns brown, then blocking with 0.3% hydrogen peroxide for 10-15 minutes is recommended, eliminating the high background fluorescence, and making the visualization of true labeling possible [3]. Like peroxidases, endogenous alkaline phosphatases can be found in the kidney, intestines, placenta, and lymph tissue, and can cause high background fluorescence when using an alkaline phosphatase fluorophore [3]. The tissue can be tested for alkaline phosphatase activity by applying BCIP/NT, and is positive if the tissue turns blue, indicating that a blocking step is needed. In this case, blocking can be completed using Levamisole and a chromogenic substrate, or in some cases, simply a weak acid block.

Biotin Blocking

If a biotin detection system is being used to detect target proteins in tissue, it is recommended that endogenous biotin sites on the tissue are blocked to avoid non-specific binding and Autofluorescence. Biotin is naturally expressed in the kidney, liver, brain, breast, and stomach tissue, as stated on The Human Protein Atlas. Tissue blocking can be done using avidin followed by biotin to block any remaining free biotin sites.

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